Reproduction in deep‐sea vent shrimps is influenced by diet, with rhythms apparently unlinked to surface production

Abstract Variations in offspring production according to feeding strategies or food supply have been recognized in many animals from various ecosystems. Despite an unusual trophic structure based on non‐photosynthetic primary production, these relationships remain largely under‐studied in chemosynthetic ecosystems. Here, we use Rimicaris shrimps as a study case to explore relationships between reproduction, diets, and food supply in these environments. For that, we compared reproductive outputs of three congeneric shrimps differing by their diets. They inhabit vents located under oligotrophic waters of tropical gyres with opposed latitudes, allowing us to also examine the prevalence of phylogenetic vs environmental drivers in their reproductive rhythms. For this, we used both our original data and a compilation of published observations on the presence of ovigerous females covering various seasons over the past 35 years. We report distinct egg production trends between Rimicaris species relying solely on chemosymbiosis—R. exoculata and R. kairei—and one relying on mixotrophy, R. chacei. Besides, our data suggest a reproductive periodicity that does not correspond to seasonal variations in surface production, with substantial proportions of brooding females during the same months of the year, despite those months corresponding to either boreal winter or austral summer depending on the hemisphere. These observations contrast with the long‐standing paradigm in deep‐sea species for which periodic reproductive patterns have always been attributed to seasonal variations of photosynthetic production sinking from the surface. Our results suggest the presence of an intrinsic basis for biological rhythms in the deep sea, and bring to light the importance of having year‐round observations in order to understand the life history of vent animals.

three congeneric shrimps differing by their diets. They inhabit vents located under oligotrophic waters of tropical gyres with opposed latitudes, allowing us to also examine the prevalence of phylogenetic vs environmental drivers in their reproductive rhythms. For this, we used both our original data and a compilation of published observations on the presence of ovigerous females covering various seasons over the past 35 years. We report distinct egg production trends between Rimicaris species relying solely on chemosymbiosis-R. exoculata and R. kairei-and one relying on mixotrophy, R. chacei. Besides, our data suggest a reproductive periodicity that does not correspond to seasonal variations in surface production, with substantial proportions of brooding females during the same months of the year, despite those months corresponding to either boreal winter or austral summer depending on the hemisphere.
These observations contrast with the long-standing paradigm in deep-sea species for which periodic reproductive patterns have always been attributed to seasonal variations of photosynthetic production sinking from the surface. Our results suggest the presence of an intrinsic basis for biological rhythms in the deep sea, and bring to light the importance of having year-round observations in order to understand the life history of vent animals.

K E Y W O R D S
biological rhythms, crustacean, deep sea, hydrothermal vent, reproduction, seasonality, trophic ecology

T A X O N O M Y C L A S S I F I C A T I O N
Life history ecology 1 | INTRODUC TI ON Relationships between feeding strategies and reproduction have been extensively studied for a number of animal phyla in various ecosystems (Almeida et al., 2018;Broderick et al., 2003;Gutiérrez et al., 2020;Lin & Shi, 2002;Meiri et al., 2012;Pierotti & Annett, 1990;Qian & Chia, 1991;Sibly et al., 2012;Tziouveli et al., 2011). However, all these studies are based on ecosystems where photosynthetic primary production is the sole basis of food webs. A number of ecosystems on Earth, such as deep-sea hydrothermal vents, cold seeps, and organic falls, rely on another primary organic source: chemosynthesis (German et al., 2011). These chemosynthesis-based ecosystems are supported by microbial communities using chemical energy from geofluids to produce their organic matter. There, animals can derive nutrition from either chemosynthesis through intricate symbiotic associations or direct consumption of free-living chemosynthetic microbes, sinking phytodetrital production from the surface, or a mixture of both, leading to the emergence of trophic networks often differing strikingly from those found elsewhere (Dubilier et al., 2008;Govenar, 2012;Sogin & Leisch, 2020). In this context, these environments are natural laboratories to study the relationships between reproduction and diet.
Although most studied species from deep-sea chemosynthesisbased ecosystems exhibit aperiodic reproductive patterns-referred to continuous in some studies-as seen in some alvinellid and polynoid polychaetes and several gastropod families (Faure et al., 2007;Jollivet et al., 2000;Marticorena et al., 2020;Tyler et al., 2008), seasonal cycles following variations in surface primary production have also been observed in many taxa including bathymodioline mussels and alvinocaridid shrimps (Copley & Young, 2006;Dixon et al., 2006;Tyler et al., 2007).
Additionally, both aperiodic and seasonal reproductive patterns may be observed in different species belonging to the same family, like in the bythograeid crabs (Hilário et al., 2009;Perovich et al., 2003).
Although the trophic strategy of these species is not always known in detail, seasonal reproduction has so far only been observed in those species capable of assimilating photosynthetic sinking particles, even if it constitutes a negligible part of their diet (Riou et al., 2010). Moreover, a clear link between surface productivity and reproductive patterns could be established in bythograeid vent crabs, with species inhabiting under highly oligotrophic waters (South Pacific Gyre) exhibiting aperiodic gametogenesis, while others living in regions with more seasonally fluctuating productivity showed corresponding reproductive patterns (Hilário et al., 2009;Perovich et al., 2003). Such examples have led to the hypothesis that the rhythm and periodicity of sinking photosynthetic matter are key time-synchronizing cues for deep-water animals exhibiting seasonal reproduction, signaling individuals to spawn at the same period of the year.
The symbiotic and congeneric shrimps Rimicaris exoculata and R. kairei are among the most iconic hot-vent animals, with unique adaptations such as hosting symbiotic bacteria in their enlarged cephalothorax cavity and the fused "dorsal eye," a novel sensory system representing a pinnacle of adaptation to vents (Zbinden & Cambon-Bonavita, 2020). The entire family they belong to, Alvinocarididae, is endemic to vents and seeps with different taxa exhibiting various trophic regimes and various levels of reliance on symbiosis (Apremont et al., 2018;Gebruk et al., 2000;Rieley et al., 1999), constituting an ideal case for elucidating relationships between reproduction and diet in chemosynthesis-based ecosystems. Species with different trophic strategies often co-occur in the same vent fields, for example, R. exoculata, which relies on symbiosis and the mixotrophic R. chacei, allowing for a straightforward comparison between congeneric species. The symbiotrophic species such as R. exoculata and R. kairei exhibit greatly inflated cephalothoraxes and live in large aggregations around the hot fluid flows (Gebruk et al., 2000;Methou et al., 2020;Rieley et al., 1999;Streit et al., 2015;Van Dover, 2002), whereas the mixotrophic species R. chacei (Apremont et al., 2018;Gebruk et al., 2000;Methou et al., 2020) lacks the inflated cephalothorax and lives in smaller groups, hidden under mussels beds or interstices between sulfides and at a greater distance from the hot fluid sources (Methou et al., 2022).
Attempts to understand the reproduction of Rimicaris shrimps have so far been inconclusive due to contradicting evidences. As with most carideans (Correa & Thiel, 2003), Rimicaris shrimps are gonochoric, with the females brooding their eggs under the abdomen until the larva hatches (Hernández-Ávila et al., 2021;Ramirez-Llodra et al., 2000).
While aperiodic reproduction was initially suggested from the examination of their reproductive tissues (Copley et al., 2007;Ramirez-Llodra et al., 2000), very few ovigerous individuals were collected in over 35 years of seagoing expeditions, despite a relatively focused and repeated sampling effort (Copley et al., 2007;Komai & Segonzac, 2008;Ramirez-Llodra et al., 2000;Shank et al., 1998;Vereshchaka, 1997;Williams and Rona, 1986). Brooding females were first found in small numbers in 2007 (Guri et al., 2012), and much greater numbers were recovered in 2014 (Hernández-Ávila et al., 2021;Methou et al., 2019), around a restricted time period between January and March. These females were living within dense aggregations close to vent orifices (Hernández-Ávila et al., 2021), refuting the previous hypothesis that brooding females would migrate to the vent periphery to protect their eggs from vent fluid, as observed for bythograeid crabs (Perovich et al., 2003) and Kiwa squat lobsters (Marsh et al., 2015). Taken together, these recent findings are suggestive of a seasonal reproductive cycle possibly linked to surface primary production, as shown in vent crabs, bathymodiolin mussels, and some other alvinocaridid shrimps (Copley & Young, 2006;Dixon et al., 2006;Tyler et al., 2007). However, the low primary productivity of oligotrophic surface waters and the resulting overall low export to the deep sea where these shrimps have been collected (−3500 m depth) (Harms et al., 2021;Pabortsava et al., 2017) challenge the plausibility of such a link.
Here, we compare reproductive outputs-that is, fecundities and egg volumes-of three congeneric vent shrimps with contrasting diets, using R. exoculata and R. kairei that are fully dependent on chemosymbiosis, and R. chacei with a mixotrophic diet combining symbiosis, bacterivory, and scavenging, to investigate whether links between diet and reproduction are also present in chemosynthetic ecosystems. The distribution of these species in two different hemispheres (Northern Atlantic for R. exoculata and R. chacei, Southern Indian Ocean for R. kairei) with similar levels of, but seasonally opposed, surface production allows us to evaluate whether their brooding periods are in synchrony with patterns of surface primary productivity. Ovigerous females included both brooding females and females that had just released their larvae but still retained modified pleopods with pieces of egg envelopes attached, characteristic of a recent brooding status (which we call "hatched broods" hereafter), following Nye et al. (2013). Ready-to-hatch broods were identified similarly, although these still had a few eggs left attached to their pleopods with clear fully developed larval structures. Broods consisted of fertilized eggs (hereafter sometimes called "eggs" for ease of language) containing developing embryos within an envelope, maintained together between the mother's modified pleopods. Within a brood, all embryos exhibited similar development stage. Embryonic stages in broods were identified and classified in three developmental stages (early, mid, and late) as seen through their transparent envelope.

| Shrimp and egg measurements
Carapace length (CL) of each female was measured with Vernier calipers from the posterior margin of the ocular shield (or eye socket) to midpoint of the posterior margin of the carapace, with an estimated precision of 0.1 mm. For each brood, the total number of eggs was manually counted. Ten eggs were selected randomly to measure maximum and minimum egg diameters. The volume of eggs was estimated following the same method as Hernández-Ávila et al. (2021), considering a spheroid volume v = 4 3 π. r 1 . r 2 , where r 1 and r 2 are half of maximum and minimum axis of each egg, respectively. Some ovigerous females harbored damaged broods where a part of the eggs was obviously missing while the remaining ones were clearly not ready to hatch, precluding natural spawning. These losses may be due to developmental abortion but most probably to sampling damages, and such broods were thus discarded from our fecundity analysis (17% of R. exoculata, 21.1% of R. chacei, and 7.1% of R. kairei broods collected). In addition, although the brooding state of females from the YK01-15 (2002)  Proportions of ovigerous females within samples were calculated against the number of sexually mature females (see Appendix S1).

| Statistical analysis
Brooding females were grouped according to sampling year, species, and vent field. Visual examination of our dataset and Shapiro-Wilk normality tests revealed that size, egg number, and egg volume of Rimicaris brooding females deviated from a normal distribution. Therefore, nonparametric tests were used for intergroup comparison, with a Kruskal-Wallis test followed by post hoc Dunn's tests when three or more groups were compared (see Appendix S1 for detailed p-values of these tests in Tables S1 and S2). Spearman's rank-order correlation was used to assess relationship between log e -transformed realized fecundity and log e -transformed carapace length of the different shrimp groups. Egg stage proportions between groups were compared using the chi-squared test with Yate's correction. All analyses were performed in R v. 4.0.3 (R Core Team, 2020).
Overall, size-specific fecundity ranged from 23 to 112 eggs mm −1 for R. kairei and from 26 to 255 eggs mm −1 for R. chacei (Figure 3b). Large differences were also observed in egg volumes between the three Rimicaris species (Kruskal-Wallis test, H = 49.80, p < .05) (Figure 3c,d). Thus, egg volumes of R. chacei were significantly smaller than those of R. exoculata at any stage across all vents (Dunn's multiple comparison test, p < .05) (Figure 3c). In contrast, differences in egg volumes were more limited between R. exoculata and R. kairei with significantly larger eggs for R. kairei from Kairei compared with R. exoculata from TAG for eggs with mid-stage embryos only (Figure 3d). Surprisingly, we did not find gradual increase in egg volumes between successive developmental stages of R. chacei as was observed in the two other Rimicaris species, either at TAG (Kruskal-Wallis test, H = 2.57, p > .05) or at Snake Pit (Kruskal-Wallis test, H = 0.61, p > .05). Of note, we did not observe ready-to-hatch or hatched egg broods in R. chacei as was seen in the other shrimps.

| Temporal variations in the presence of Rimicaris ovigerous females
A large number of ovigerous females of R. exoculata and R. chacei were retrieved in February 2018 at both MAR vent fields (Figure 4a).
Indeed, ovigerous females represented 55.4% and 18.2% of all the sexually mature R. exoculata females collected respectively at TAG and Snake Pit and 70.6% or 32.3% of all the sexually mature R. chacei females, respectively, at TAG and Snake Pit. Ovigerous females of R. exoculata were also present between late March and April 2017 although in a relatively lower proportion, constituting 25.7% and 4% of sexually mature females collected at TAG and Snake Pit, respectively ( Figure 4a). The majority of R. exoculata ovigerous females were collected in dense aggregations close to vent orifices with only two ovigerous females collected outside these assemblages. On the contrary, ovigerous females of R. chacei were collected either in visually "hidden" aggregations under mussels beds or sulfide block interstices where large populations of their adults have been retrieved (Methou et al., 2022), or within dense aggregations of R. exoculata.

| DISCUSS ION
Altogether, our results show that Rimicaris exoculata and R. kairei had much lower size-specific fecundities and much larger egg volumes than R. chacei. The realized fecundity correlated positively with carapace length in all three species, although the relationship between the two variables was distinct in R. chacei compared with the two other species. Spawning appears to be seasonal in all three species, occurring between January and April, possibly cued to optimal larval release conditions. Surprisingly, the data we present suggest a similar spawning season for R. kairei and the other two species, despite their experience of opposite primary production trends as they were collected at opposing latitudes in two different hemispheres.

| Distinct reproductive strategies in Rimicaris shrimps with evidence of diet influence
Rimicaris exoculata and Rimicaris chacei exhibit distinct reproductive traits unlikely to simply result from differences in mean body sizes.
Rimicaris kairei is similar to R. exoculata, in terms of size-specific fecundity and mean egg size. Our results indicate that the two groups (R. exoculata and R. kairei, vs R. chacei) are situated at two ends of the trade-off between size-specific fecundity and egg sizes currently observed within alvinocaridids (Copley & Young, 2006;Nye et al., 2013;Ramirez-Llodra et al., 2000;Ramirez-Llodra & Segonzac, 2006). Rimicaris chacei brooding females display one of the highest size-specific fecundities and one of the smallest mean egg volumes among alvinocaridids, whereas R. exoculata and R. kairei brooding females present a rather low size-specific fecundity and a large mean egg volume for this family. In addition, annual comparison of R. exoculata reproductive traits also revealed a stable sizespecific fecundity over the years, but some levels of variation were seen in the mean egg volume among the sampling years.
These variations between species mirror differences observed in benthic post-settlement stages of these two shrimp groups, with R. chacei exhibiting significantly smaller body size for settled juveniles, as well as much larger relative proportions of juveniles compared with adults in populations (Methou et al., 2020(Methou et al., , 2022. These previous works on the population demographics of the two MAR species showed a drastic post-recruitment collapse in R. chacei with a high juvenile mortality, hypothesized to be the result of a combination of factors, including interspecific competition between juveniles of the two congeneric species and size-specific predation of the surrounding fauna on R. chacei juveniles (Methou et al., 2022). Such a collapse was not observed in R. exoculata populations, which exhibited a larger proportion of adults than juveniles, potentially close to the carrying capacities of their vent field. These differences in terms of age-and size-specific selection acting heterogeneously on the different life stages of the two species could be the source of their niche partitioning (Methou et al., 2020(Methou et al., , 2022 and lead to the different life history strategies observed herein for these species.
Furthermore, density-dependent selection could be a driving factor, where R. exoculata is always nearing the equilibrium population, and therefore invest more in each offspring (Methou et al., 2020(Methou et al., , 2022, while R. chacei has less stable populations and therefore produces more offsprings. The integration in this theoretical framework of R. hybisae, the sister species of R. chacei from vents on the Mid Cayman Spreading Centre, phylogenetically close but behaviorally distinct, is interesting as size-specific fecundity of R. hybisae is more similar to that of R. exoculata and R. kairei rather than R. chacei (Nye et al., 2013). Rimicaris hybisae share with R. exoculata/R. kairei both a comparable ecological context, living in dense aggregations close to vent fluids emissions, and a comparable gross morphology, with an enlarged cephalothorax (Zbinden & Cambon-Bonavita, 2020), characters lacking in R. chacei. These are linked to distinct feeding strategies between the two groups of shrimps, with a mixotrophic diet for R. chacei (Apremont et al., 2018;Gebruk et al., 2000;Methou et al., 2020) and dependency on chemosynthetic symbionts for others including R. hybisae (Gebruk et al., 2000;Methou et al., 2020;Rieley et al., 1999;Streit et al., 2015;Van Dover, 2002). Similar to R. exoculata and R. kairei, R. hybisae is also extremely abundant where it occurs and the adult population is potentially nearing the carrying capacity. We therefore posit that the differences we observe between fecundities in our three Rimicaris shrimps are likely linked to their feeding ecology (and occupied niche), rather than being constrained by their phylogeny.

F I G U R E 3 Comparison of reproductive features between
Despite variations in average egg number, mean egg sizes of R. hybisae and R. chacei were in contrary comparable with similar maximum and minimum egg diameters (Nye et al., 2013). Such similarities on mean egg sizes were more unexpected and would deserve further work, including additional alvinocaridid shrimp species with different feeding strategies to elucidate whether egg sizes could be influenced by feeding strategies or by other factors such as phylogeny. A potential observer effect, as shown here for egg volumes of R. exoculata collected during the BICOSE expedition, cannot be excluded either.  Figure 4a), these results support the existence of a brooding period for R. exoculata mostly during boreal winter, starting in January-February and ending around March-April. Moreover, we did not observe significant variability in reproductive features of R. exoculata, either in terms of fecundity or proportions of reproductive females, between different sampling years-indicating a relative interannual stability of these traits (Figure 2). Since gamete production can be sustained continuously in species depending on local chemosynthesis for their diet, mechanisms driving periodicity in shrimp life cycles are more likely to act on their planktotrophic larval phase through seasonality in availability of photosynthetically derived food (Eckelbarger and Watling, 1995). The few previous studies looking at a limited number of samples indeed reported asynchronous ovarian development for R. exoculata, taken to suggest aperiodic egg production (Copley et al., 2007;Ramirez-Llodra et al., 2000). However, the maximum oocyte size appeared to be lower in individuals collected in summer than in those collected in autumn, regardless of the vent site (Hernández-Ávila et al., 2021;Ramirez-Llodra et al., 2000). Thus, this may result from a gradual size increase in mature oocytes in ovaries, culminating in winter, prior to spawning. Furthermore, oogenesis in Rimicaris might also be distinct from spawning activity, with mature ovaries retained for extended periods until the seasonal spawning, as observed in some deep-sea echinoderms (Eckelbarger & Watling, 1995), followed by brooding and larval hatching.
Large proportions of R. chacei ovigerous females were also retrieved in February 2018 at the two vent fields, in contrast to historical sampling at other periods along the MAR (Figure 4b). Hence, a similar periodicity in reproduction could exist for both Rimicaris shrimps at MAR. Ovigerous R. chacei females were, however, scarce in other expeditions both during and outside this period (Figure 4b), which might be due to difficulties in sampling this rather fast swimming species, thus preventing so far, a full appreciation of its reproductive activity. A similar seasonal pattern was also suggested for the sister species of R. chacei, R. hybisae, although based on a limited dataset (Nye et al., 2013). Taken alone, these results on north MAR Rimicaris populations seemed to indicate aperiodic gametogenesis coupled with seasonal spawning, synchronized by the peak of photosynthetic production sinking from surface, as reported in other deep-sea animals with seasonal reproductive periodicity (Dixon et al., 2006;Perovich et al., 2003;Tyler et al., 2007). This pattern was unexpected given their distribution in different hemispheres, at vent fields with opposite latitudes (Figure 1a) where these shrimps are expected to experience opposite trends in terms of surface primary production along the year (Harms et al., 2021;Pabortsava et al., 2017), driving opposite reproductive timing. Although our dataset for R. kairei is more limited than for R. exoculata, our results show that the two sister species share the same periodic brooding cycle, regardless of local seasonal variations in surface photosynthetic productivity, which also appear to be relatively weak in their respective regions (Harms et al., 2021;Pabortsava et al., 2017).
These observations cannot be generalized to the entire Alvinocarididae family, however, as evidences of potential aperiodic brooding have been published for some other species in the family. Regular sampling of Mirocaris fortunata, for example, yielded ovigerous females throughout the year (Komai & Segonzac, 2003;Methou, 2019;Ramirez-Llodra et al., 2000) and similarly in the sampling records of Rimicaris variabilis (Komai & Tsuchida, 2015;Komai et al., 2016). It therefore appears unlikely that phylogenetic constraints could have maintained periodic brooding in some alvinocaridids but not in others (Figure 1c). Then again, some alvinocaridid species may also be influenced by seasonality in the surface production, like Alvinocaris stactophila that inhabit relatively shallow cold seeps under highly productive coastal waters and exhibit a brooding period between November and March (Copley and Young, 2006), matching the surface productivity. Hence, several mechanisms relating reproductive rhythms and environmental variability may coexist in vents and other deep-sea ecosystems, even within the same family of animals.
Besides light and food supply, temperature has also been recognized as an important external timing cue in marine organisms (Mat, 2019) but observations from long-term observatories deployed in a few vent fields did not reveal any seasonal variations in temperature at an annual scale (Barreyre et al., 2014;Cuvelier et al., 2017). Moreover, hydrothermal vent ecosystems, compared to most of the deep sea where temperature is relatively constant within regions, exhibit heat anomalies with steep and unpredictable gradients at small spatial scales even within the same animal aggregation (Schmidt et al., 2008). In the current state of our knowledge, the synchronizing factors driving reproductive cycles in Rimicaris shrimps from the MAR and the CIR remain unclear. Recently, it was revealed that bathymodiolin mussels inhabiting vents on the MAR retain and express circadian clock genes following tidal cycles, which may be mediated by either tidal stimulus or an internal clock (Mat et al., 2020). It is not completely out of the question that the Rimicaris shrimp species studied herein may also use a set of internal biological clocks to time their reproductive activity.
Overall, even if primary surface production may be a key synchronizing cue for some deep-sea species, as demonstrated in bathymodiolin mussels and vent crabs (Dixon et al., 2006;Tyler et al., 2007), our results underscore that reproductive cycles and seasonality in the deep sea are not as simple as it was once thought to be. A thorough understanding of the reproductive ecology of the dominant species shaping the ecosystem is key to assessing the resilience of deep-sea ecosystems, but remain incomplete so far even for emblematic taxa such as Rimicaris. With deep-sea mining for massive sulfides and other resources being imminent (Van Dover et al., 2018), the habitat of these unique animals that can live nowhere else is being threatened. Research expeditions in a particular region tend to be conducted in similar timings of the year due to seasonal variations in sea and weather conditions, but our results show that communities need to be revisited at different times of the year.
Elucidating the ecology of these species is now more important than ever, and our data exemplify the importance of time-series studies.

ACK N OWLED G M ENTS
The authors thank the captains and crews of R/V Pourquoi pas?
and HOV Nautile submersible team for their efficiency, as well as the chief scientists and scientific parties of the HERMINE (chief scientist: Yves Fouquet; https://doi.org/10.17600/ 17000200) and BICOSE 2 (chief scientist: Marie-Anne Cambon-Bonavita; https:// doi.org/10.17600/ 18000004) cruises. Further thanks also go to the captains and crews of the R/V Yokosuka, and the HOV Shinkai 6500 submersible team for their continuous support of the scientific activity at sea, as well as the chief scientists and scientific parties during the research cruises YK01-15 (chief scientist: Ken Takai), YK09-13 (chief scientists: Kentaro Nakamura, Satoshi Nakagawa), and YK16-E02 (chief scientist: Ken Takai). We also thank Dr. Ivan Hernández-Ávila (Facultad de Ciencias Naturales, Universidad Autónoma del Carmen) for his help sorting the specimens onboard and the analyses to determine the sexual maturity of these shrimps.

CO N FLI C T O F I NTE R E S T
The authors declare they have no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Dataset used within this manuscript can be accessed through the Ifremer SEANOE (SEA scieNtific Open data Edition) database at: https://doi.org/10.17882/ 84195.